Overall Abstract of the Program Endothelial barrier dysfunction is a central factor in the pathogenesis of protein-rich lung edema formation and lung inflammation, the hallmarks of acute respiratory distress syndrome (ARDS). Due to the complexity of these conditions, attempts to develop drugs and cell-based therapies have thus far not been successful. Thus, whereas previously explored therapeutics specifically targeted the complex multistep process that occurs during the lung injury phase, here we propose to focus on processes underlying the resolution of vascular leakage and pulmonary edema. The central hypothesis of the Program Project application for years 16 to 20 is that resealing of the endothelial barrier at the level of adherens junctions (AJs) is required to restore junctional integrity, and thereby to normalize lung fluid balance. The research focus in all Projects will thus be on delineating the role of overlapping but specialized signaling pathways that operate to re-seal AJs, and on this basis, identifying novel therapeutic targets that can be harnessed to reverse the course of the disease. In Project 1, Dr. Asrar Malik, PI, will test the novel hypothesis that restoration of lung endothelial barrier is regulated by two complementary mechanisms, the vascular endothelial protein tyrosine phosphatase (VE-PTP)-mediated stabilization of VE-cadherin, and the Rac1-mediated formation of adhesive bonds between VE-cadherin molecules at AJs. These studies will define the signaling mechanisms activated by VE-PTP interaction with VE-cadherin, and with tyrosine kinase receptor Tie2 in regulating the integrity of the lung endothelial barrier. In Project 2, Dr. Jalees Rehman, PI, will test the central hypothesis that inflammation-induced glycolysis in lung endothelial cells (ECs) is an intrinsic mechanism necessary for restoration of endothelial barrier function. Studies will examine the roles of the key rate-limiting kinase in regulating glycolysis 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFK-FB3), and activation of glutamine metabolism in mitochondria in endothelial barrier repair. In Project 3, Dr. Dolly Mehta, PI, will test the fundamental hypothesis that S1P receptor-1 (S1PR1) expression regulates lung vascular barrier integrity, and is essential for restoring lung fluid balance. These studies will define mechanisms controlling the cell surface expression of S1PR1 in ECs following lung injury, and the role of interaction of S1PR1 with the VEGF receptor (VEGFR2) in establishing vascular endothelial integrity. In Project 4, Dr. Viswanathan Natarajan, PI, will test the hypothesis that spatial and coordinated production of lipid second messengers such as phosphatidic acid by phospholipase D2 (PLD2) and of S1P by sphingosine kinase 1 (SphK1) is required for lamellipodia formation and recycling of VE-cadherin to AJs to restore the EC barrier. These studies will define the function of these bioactive lipids as key mediators inducing the formation of AJs, and thereby the endothelial barrier integrity and how their function can be optimized to restore lung-fluid balance. We posit that this concerted effort using novel and rigorous approaches described in each project of the Program, will lay new conceptual groundwork for identifying novel therapeutic targets to treat ARDS.